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Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides.


ABSTRACT: The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/?1 = 1.3 ± 0.6 nM, and 1/?2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.

SUBMITTER: Al-Ani G 

PROVIDER: S-EPMC4100786 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides.

Al-Ani Gada G   Briggs Koan K   Malik Shuja Shafi SS   Conner Michael M   Azuma Yoshiaki Y   Fischer Christopher J CJ  

Biochemistry 20140630 27


The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and  ...[more]

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